Immunoregulatory protein B7-H3 promotes growth and decreases sensitivity to therapy in metastatic melanoma cells.

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Immunoregulatory protein B7-H3

promotes growth and decreases sensitivity to therapy in metastatic melanoma cells

Karine Flem-Karlsen | Christina Tekle | Yvonne Andersson | Kjersti Flatmark | Øystein Fodstad |

Caroline E. Nunes-Xavier

Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr

DOI: 10.1111/pcmr.12599

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Pigment Cell Melanoma Res. 2017;1–10. wileyonlinelibrary.com/journal/pcmr |₁

Received: 16 January 2017

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Accepted: 9 May 2017 DOI: 10.1111/pcmr.12599

O R I G I N A L A R T I C L E

Immunoregulatory protein B7- H3 promotes growth and

decreases sensitivity to therapy in metastatic melanoma cells

Karine Flem-Karlsen

^1,2

| Christina Tekle

¹

| Yvonne Andersson

¹

| Kjersti Flatmark

^1,2

| Øystein Fodstad

^1,2

| Caroline E. Nunes-Xavier

¹

1Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway

2Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Correspondence

Caroline E. Nunes-Xavier Email: [email protected] Funding information

The Norwegian Cancer Society; The Research Council of Norway, Grant/Award Number:

239,813

Summary

B7- H3 (CD276) belongs to the B7 family of immunoregulatory proteins and has been implicated in cancer progression and metastasis. In this study, we found that metastatic melanoma cells with knockdown expression of B7- H3 showed modest decrease in proliferation and glycolytic capacity and were more sensitive to dacarbazine (DTIC) chemotherapy and small- molecule inhibitors targeting MAP kinase (MAPK) and AKT/mTOR pathways: vemurafenib (PLX4032; BRAF inhibitor), binimetinib (MEK- 162; MEK inhibitor), everolimus (RAD001; mTOR inhibitor), and triciribidine (API- 2; AKT inhibitor).

Similar effects were observed in melanoma cells in the presence of an inhibitory B7- H3 monoclonal antibody, while the opposite was seen in B7- H3- overexpressing cells.

Further, combining B7- H3 inhibition with small- molecule inhibitors resulted in significantly increased antiproliferative effect in melanoma cells, as well as in BRAF^V600E mutated cell lines derived from patient biopsies. Our findings indicate that targeting B7- H3 may be a novel alternative to improve current therapy of metastatic melanoma.

K E Y W O R D S

AKT/mTOR inhibitors, B7-H3/CD276, MAPK, melanoma, targeted therapy, vemurafenib

1 | INTRODUCTION

The only chemotherapeutic Food and Drug Administration (FDA)- approved drug for treatment of malignant melanoma is the alkylat- ing agent dacarbazine (DTIC), although its efficacy is modest (Bhatia, Tykodi, & Thompson, 2009). In BRAF- mutated melanomas, targeted therapy has improved survival of patients with advanced metastatic melanoma (Eggermont & Robert, 2011; Vennepureddy, Thumallapally, Motilal Nehru, Atallah, & Terjanian, 2016). BRAF^V600 is mutated in ap- proximately 45% of cutaneous and 10% to 20% of mucosal or acral melanomas (Goldinger, Murer, Stieger, & Dummer, 2013; Karachaliou et al., 2015), with other commonly mutated or amplified genes being NRAS and c- KIT, and less frequently HRAS (Albino, Le Strange, Oliff, Furth, & Old, 1984). Thus, new alternative therapeutic alternatives are needed for melanoma patients lacking BRAF mutation. Resistance to targeted therapy is a major issue in melanoma, usually correlated

with reactivation of MAPK and increase in AKT activation. Hence, inhibitors of MEK and the PI3K/AKT/mTOR pathway are being tested clinically in combination with other inhibitors (Ascierto et al., 2013;

Fedorenko, Gibney, Sondak, & Smalley, 2015; Greger et al., 2012;

Thumar, Shahbazian, Aziz, Jilaveanu, & Kluger, 2014). Further, the newly approved immunotherapies using PD- 1 and CTL- 4 checkpoints inhibitors (Ott, Hodi, & Robert, 2013; Pardoll, 2012) have shown great promise in the clinic, as has blocking PD- 1 in combination with its li- gand B7- H1/PD- L1 (Dossett, Kudchadkar, & Zager, 2015; Mahoney, Freeman, & Mcdermott, 2015).

B7- H1/PD- L1 is a member of the B7 family of proteins and is frequently overexpressed in melanoma (Kakavand et al., 2015).

Proteins in the B7 family are important immune response regula- tors (Flies & Chen, 2007) and can mediate metastasis- related sig- nals and support tumor development (Leung & Suh, 2014). Another family member, B7- H3, is also highly expressed in melanoma cells This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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and implicated in tumor immunity (Chapoval et al., 2001; Loo et al., 2012; Tekle et al., 2012; Wang, Chong et al., 2013; Xu, Cheung, Guo, & Cheung, 2009; Yi & Chen, 2009). Subcellular localization of B7- H3 is important for exerting its function, and it is predomi- nantly found in the cell membrane and cytoplasm, but also in the nucleus, of tumor cells (Chen, Tekle, & Fodstad, 2008; Ingebrigtsen et al., 2014; Liu et al., 2013; Wang, Zhang et al., 2013). Additionally, B7- H3 has been detected in exosomes, and as soluble isoforms in serum (Chen et al., 2013; Kshirsagar et al., 2012; Marimpietri et al., 2013; Zhang et al., 2008).

B7- H3 affects sensitivity to various drugs and targeted therapies in several cancer types (Jiang, Liu, Liu, Zhang, & Hua, 2016;

Liu et al., 2011; Nunes- Xavier et al., 2016; Zhang et al., 2015a,b;

Zhao et al., 2013), but has not been addressed in malignant melanoma. In this study, we have assessed the role of B7- H3 in the sensitivity of melanoma cells to the chemotherapeutic agent DTIC, and to clinically relevant MAPK and AKT/mTOR inhibitors: vemurafenib, binimetinib, everolimus, and triciribidine. We found that low expression or inhibition of B7- H3 renders the cells more sensitive to these drugs in addition to decreasing their glycolytic capacity. Our results suggest that targeting B7- H3 may be a novel supplement to improve current anticancer therapies in metastatic melanoma.

2 | RESULTS

2.1 | B7- H3 promotes growth and glycolysis of melanoma cells

To study the role of B7- H3 in melanoma cell growth, we used FEMX- 1 and SKMEL- 28 cells with knockdown or overexpressed protein levels

Significance

The treatment of metastatic melanoma has experienced a shift in the past years with vemurafenib, targeting mutated BRAF, and the rise of immunotherapy. However, only a portion of patients responds to the treatment and the rate of relapse is high.

Thus, new targeted therapy is urgently needed for metastatic melanoma patients. We found that reducing or inhibiting the expression of B7- H3 in metastatic melanoma cells reduced cell growth and glycolytic capacity, and increased sensitivity to chemotherapy and various targeted therapies. Our findings indicate that targeting B7- H3 may be a novel alternative to improve current therapy of metastatic melanoma.

F I G U R E 1 Role and localization of B7- H3 in FEMX- 1 melanoma cells. (a) Immunoblot of B7- H3 and tubulin expression from total lysates from FEMX- 1 shSCR and shB7- H3 cells and FEMX- 1 vector and B7- H3- overexpressing cells. shSCR, control short hairpin scrambled cells;

shB7- H3, short hairpin B7- H3 cells; VEC, control vector cells; B7- H3, overexpressing B7- H3 cells. Plots show quantified immunoblot bands from B7- H3/tubulin in arbitrary units (A.U.) from three independent experiments ± S.E.M. (b) Immunoblot from total cell lysates and exosome fractions from FEMX- 1 control (vector) and B7- H3- overexpressed cells (B7- H3). Plots show quantified immunoblot bands from B7- H3/CD63 from three independent experiments ± S.E.M. (c) Relative proliferation of cells variants as in A. Left panels: Average relative proliferation of three independent experiments was measured by MTS assay after 3 days in culture ± S.E.M. Right panels: Cell confluence was measured growing the cells in IncuCyte FLR or IncuCyte ZOOM Kinetic Imaging System (Essen BioScience), ± S.D. Cells were scanned every 3 hr during the times indicated. (d) Colony formation of cell variants as in A ± S.D. (e) Seahorse Extracellular Flux Analyzer XF96e was used to measure the extracellular acidification rate (ECAR) and in FEMX- 1 shSCR and shB7- H3 cells ± S.D. Note significant reduction in glycolytic capacity in FEMX- 1 shB7- H3 cells compared with shSCR cells. Statistically significant results (p < .05) are marked with*

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of B7- H3 (Figures 1a and 2a; (Tekle et al., 2012)). As different subcellular localizations of B7- H3 have been reported, this was examined.

B7- H3 was found mainly in the cell membrane, with a weaker protein staining in the cytoplasm (Figure S1, left panels). We did not detect any nuclear staining. In FEMX- 1 cells, B7- H3 was co- localized with LAMP2 and CD63 (Figure S1, middle panels), indicating that B7- H3 is also expressed in intracellular membrane- bound vesicles, that is, lysosomes and late endosomes. Co- localization was not found with melan- A in the pigmented SKMEL- 28 cells (Figure S1, right panels), indicating that B7- H3 is not present in melanosomes. B7- H3 was also detected in exosome extracellular vesicles (Figure 1b). Notably, exosomes from the B7- H3- overexpressing cell lines had a higher expression of B7- H3 compared to empty vector cells, indicating that higher B7- H3 expression leads to more sorting of the protein to extracellular vesicles.

A modest decrease in cell proliferation and cell confluence was observed in FEMX- 1 and SKMEL- 28 B7- H3 knockdown cells (shB7- H3) compared with the control cells (shSCR) (Figure 1c, top panels, and Figure 2a, middle panels), whereas the opposite was observed in B7- H3- overexpressing FEMX- 1 cells (Figure 1c, lower panels). Colony formation was also significantly decreased in FEMX- 1 shB7- H3 cells (Figure 1d). Recent findings link B7- H3 expression to increased Warburg effect in breast cancer cells (Lim et al., 2016; Nunes- Xavier et al., 2016). Hence, we analyzed glycolysis by measuring extracellular

acidification rate (ECAR) upon B7- H3 knockdown in two different melanoma cell lines (Figures 1e and 2a, right panel). shSCR and shB7- H3 cell variants had similar ECAR levels in basal and in glucose- induced glycolysis. However, shB7- H3 cells, in contrast to shSCR cells, did not respond to the addition of the oxidative phosphorylation inhibitor oligomycin, thus failing to induce the full glycolytic capacity of the cells.

This suggests that the cell glycolytic reserve is lower in B7- H3 knockdown cells. Similar ECAR profile to that of B7- H3 knockdown was found in the presence of an inhibitory anti- B7- H3 monoclonal antibody (α- B7- H3, BRCA84D) (Figure S2). This shows that inhibiting B7- H3 expression in melanoma cells leads to decreased Warburg effect.

Expression of B7- H3, phospho- ERK1/2 (pERK1/2), ERK1/2, phospho- AKT (pAKT), and AKT was compared in four different melanoma cell lines: FEMX- 1 (BRAF wt, HRAS^G12V mutated), SKMEL- 28 (BRAF^V600E mutated, RAS wt), HHMS (BRAF wt, RAS wt), and MeWo (BRAF wt, RAS wt) by immunoblot analysis. High expression of B7- H3, as well as expression of activated ERK1/2 and AKT, was detected in all parental cells (Figure 2b). The presence of α- B7- H3 (BRCA84D) caused a significant and antiproliferative effect in all four cell lines, as measured by cell viability and confluence (Figure 2c and Figure S3). We also observed same antiproliferative effect in WM1366 (BRAF wt, NRAS^Q61L) and WM902b (BRAF^V600E mutated, RAS wt) melanoma cells in the presence of an α- B7- H3 (22F2) (Figure 2d). This is consistent with the effect of shB7- H3 described above.

F I G U R E 2 Immunoblot and proliferation analysis of melanoma cell lines. (a) Left panel: Immunoblot of B7- H3 and tubulin expression from total lysates from SKMEL- 28 shSCR and shB7- H3 cells. Plots show quantified immunoblot bands from B7- H3/tubulin, in arbitrary units (A.U.) from three independent experiments ± S.E.M. Middle panels: Relative proliferation and cell confluence of SKMEL- 28 shSCR and shB7- H3 cells. Average relative proliferation and cell confluence of three independent experiments were measured by the MTS assay after 3 days in culture or by growing the cells in IncuCyte FLR or IncuCyte ZOOM Kinetic Imaging System (Essen BioScience) ± S.D. Results are shown from one representative experiment. Right panel: ECAR was measured by the Seahorse Extracellular Flux Analyzer XF96e in SKMEL- 28 shSCR and shB7- H3 cells ± S.D. Note significant reduction in glycolytic capacity in SKMEL- 28 shB7- H3 cells compared with shSCR cells. (b) Immunoblot of B7- H3, tubulin, pERK1/2, ERK1/2, pAKT, and AKT expression from total lysates from FEMX- 1, SKMEL- 28, HHMS, and MeWo melanoma cells. Plots show quantified immunoblot bands from B7- H3/tubulin, pERK/ERK, and pAKT/AKT A.U. from two independent experiments ± S.E.M. (c, d) Relative proliferation was measured in FEMX- 1, SKMEL- 28, HHMS, and MeWo melanoma cells in the presence of 100 ng/ml B7- H3 inhibitory antibody (α- B7- H3, BRCA84D) (c), or in WM1366 and WM902b melanoma cells in the presence of 10 μg/ml B7- H3 inhibitory antibody (α- B7- H3, 22F2) (d). Results are shown from one representative experiment. Statistically significant results (p < .05) are marked with*

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2.2 | B7- H3 knockdown and inhibition increase the sensitivity of FEMX- 1 melanoma cells to DTIC chemotherapy and MEK and AKT/mTOR inhibitors

The role of B7- H3 in FEMX- 1 (HRAS^G12V mutated) cells on drug sensitivity was studied by measuring cell confluence and proliferation of FEMX- 1 shSCR and shB7- H3 cells to DTIC chemotherapy and to targeted therapy with the MEK inhibitor binimetinib. FEMX- 1 shB7- H3 cells were more sensitive than shSCR cells to DTIC (2.01 ± 0.29- fold) and more sensitive to binimetinib (1.34 ± 0.08- fold) (Figure 3a, and Figures S4A and S4B). Additionally, the antiproliferative effect of binimetinib treatment was further increased by the simultaneous presence of α- B7- H3 (1.57 ± 0.30- fold) (Figure 3b). In accordance with this, in B7- H3- overexpressing FEMX- 1 cells, the increased B7- H3 expression led to reduced sensitivity to binimetinib (0.84 ± 0.04- fold) (Figure 4b). Immunoblot analysis of pERK1/2 and ERK1/2 in FEMX- 1 shSCR and shB7- H3 cells treated with single agents indicated a differential and independent growth inhibitory effect of DTIC with no significant change in pERK1/2 activation, whereas in binimetinib- treated cells, an expected inhibitory effect on pERK1/2 activation was seen (Figure 3c). FEMX- 1 shSCR and shB7- H3 cell variants treated with the combination of DTIC and binimetinib showed an increased growth inhibition in the FEMX- 1 shSCR cells (Figure 3a), but not in shB7- H3 cells. This suggests that

B7- H3 affects drug sensitivity through common effectors of both agents.

We also tested FEMX- 1 cell sensitivity to the mTOR and AKT inhibitors, everolimus and triciribidine. FEMX- 1 shB7- H3 cells, as well as FEMX- 1 parental cells in the presence of α- B7- H3, were more sensitive to everolimus (1.17 ± 0.11- fold and 1.62 ± 0.41- fold, respec- tively) (Figure 4a, c) and to triciribidine (Figures S5A, S5B, and S5C) than control cells. In contrast, B7- H3- overexpressing cells were less sensitivity to everolimus (0.89 ± 0.04- fold) (Figure 4b). Moreover, the parental HHMS and MeWo (BRAF wt and RAS wt) cells also showed increased sensitivity to both binimetinib and everolimus in the presence of B7- H3 antibody (Figure 4d, e). That the combination of these two inhibitors did not increase growth inhibition in the FEMX- 1 shSCR and vector control cells suggests a similar mechanistic action of the inhibitors on the growth inhibition (Figure 4a, b).

2.3 | B7- H3 knockdown and inhibition affect sensitivity of SKMEL- 28 melanoma cells to vemurafenib, binimetinib, and everolimus

The role of B7- H3 in SKMEL- 28 (BRAF^V600E mutated) cell sensitivity to the BRAF inhibitors vemurafenib, binimetinib, and everolimus was studied using cell confluence and proliferation assays. shB7- H3 cells were more sensitive to all three inhibitors as compared to control F I G U R E 3 Proliferation of FEMX- 1 shB7- H3 cells treated with DTIC and MEK inhibitor. (a, b) Relative proliferation of the cells was measured by cell proliferation (MTS) assay after 3 days of treatment of cell variants FEMX- 1 shSCR and shB7- H3 cells with 5 μg/ml DTIC, 1 μ^m binimetinib, and combination of 5 μg/ml DTIC and 1 μ^m binimetinib (a), or in parental FEMX- 1 with or without 0.1 μ^m binimetinib with or without the presence of 100 ng/ml B7- H3 inhibitory antibody (α- B7- H3, BRCA84D) (b). In all experiments, DMSO was used as a vehicle control. All MTS data are average of three independent experiments ± S.E.M., normalized, and relative to untreated cells. Statistically significant results (p < .05) are marked with *. (c) Immunoblot of B7- H3, pERK1/2, ERK1/2, and tubulin expression from total lysates from FEMX- 1 shSCR and shB7- H3 cells treated for 2 hr with 50 μg/ml DTIC, 1 μ^m binimetinib, and combination of both. Plots show quantified immunoblot bands from B7- H3/

tubulin and pERK/ERK, in arbitrary units (A.U.) from two independent experiments ± S.E.M (a)

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shSCR cells (Figure 5a, b, and Figures S6A and S6B). A significant increased sensitivity of SKMEL- 28 cells to binimetinib (1.19 ± 0.02- fold), vemurafenib (1.11 ± 0.02- fold), and everolimus (1.13 ± 0.07- fold) inhibitors was also seen in the presence of α- B7- H3 antibody (Figure 5c). As opposed to the results for FEMX- 1 cells, an additional inhibitory effect of combination treatment of binimetinib together with everolimus, as well as for the combination of vemurafenib and everolimus, was observed for SKMEL- 28 cells (Figure 5a, b).

2.4 | Targeting B7- H3 in patient- derived BRAF

^V600E

mutated metastatic melanoma cell lines

In Patient 1- , Patient 3- , and Patient 4- derived cell lines (BRAF^V600E mutated, RAS wt), we observed activation of ERK1/2 and AKT as well as high expression of B7- H3 protein (Figure 6a). All cell lines responded well to the α- B7- H3 treatment (Figure 6b, c), but showed a clear heterogeneity in response to treatment with the targeted therapies. Interestingly, Patient 1- derived cell line, which had the highest expression of B7- H3, was less sensitive to all targeted therapies, but responded to α- B7- H3, although less than the other two cell lines (Figure 6c). These differences are likely due to other cell type- specific factors. In all three patient- derived cell lines, we observed a weak yet significant additive antiproliferative effect of α- B7- H3 treatment in combination with vemurafenib and binimetinib. In cell lines from patients 1 and 4, we also observed a significant additive antiproliferative effect of α- B7- H3 in combination with everolimus (Figure 6c). Taken together, these results suggest that targeting of B7- H3 alone, or in combination with current anticancer therapies, could be a suitable alternative for treatment of B7- H3- positive malignant melanomas.

3 | DISCUSSION

B7- H3 exerts a pro- oncogenic role in cancer cells, is implicated in tumor immunology (Nygren, Tekle, Ingebrigtsen, & Fodstad, 2011;

Wang, Kang, & Shan, 2014), and has been proposed as target in many types of solid cancer (Picarda, Ohaegbulam, & Zang, 2016). In accordance with this, we found an antiproliferative effect on various melanoma cell lines by B7- H3 knockdown by short hairpin RNAs as well as upon treatment with a monoclonal antibody targeting B7- H3. Currently, targeting B7- H3 by the use of an antibody (enoblitu- zumab, MGA271, MacroGenics; (Loo et al., 2012)) is being tested in phase I clinical trials in patients with B7- H3- positive cancers, includ- ing melanoma, as single agent (ClinicalTrials.gov: NCT01391143, NCT01918930), or in combination with checkpoint inhibitors for CTLA- 4 (ipilimumab; NCT02381314) or PD- 1 (pembrolizumab;

NCT02475213).

We have previously reported that breast cancer cells with knockdown of B7- H3 showed increased sensitivity to paclitaxel chemotherapy and to AKT/mTOR inhibitors (Liu et al., 2011; Nunes- Xavier et al., 2016). Here, we show that low expression or inhibition of B7- H3 in melanoma cells renders them more sensitive to DTIC and to the inhibitors vemurafenib, binimetinib, everolimus, and triciribidine, targeting either MAPK or AKT/mTOR pathway. To our knowledge, this is the first time B7- H3 expression is linked to regulation of the sensitivity to both vemurafenib and binimetinib. Additionally, vemurafenib- resistant patient biopsy melanoma cell lines responded well to inhibition of B7- H3, as well as to binimetinib and everolimus. However, the Patient 1- derived cell line showed weaker response to α- B7- H3 therapy compared to the other patient- derived cell lines despite having high B7- H3 expression levels. This is in agreement with our observation that this F I G U R E 4 Proliferation of shB7- H3- or B7- H3- overexpressing melanoma cells treated with mTOR and MEK inhibitors. Relative proliferation of the cells was measured by MTS assay after 3 days of treatment of FEMX- 1 shSCR and shB7- H3 cells with 0.1 μ^m binimetinib, 200 nm

everolimus, and combination of 0.1 μ^m binimetinib and 200 nm everolimus (a), or in FEMX- 1 vector and B7- H3 cells with 0.1 μ^m binimetinib, 200 nm everolimus, and combination of 0.1 μ^m binimetinib and 200 nm everolimus (b), or in parental FEMX- 1 cells with or without 200 nm

everolimus with or without the presence of 100 ng/ml B7- H3 monoclonal inhibitory antibody (α- B7- H3, BRCA84D). Note: control and α- B7- H3 are the same as in Figure 3b (c), or in HHMS cells with or without 0.1 μ^m binimetinib or 200 nm everolimus with or without the presence of 100 ng/ ml α- B7- H3 (d), or in MeWo cells with or without 0.1 μ^m binimetinib or 200 nm everolimus with or without the presence of 100 ng/ml α- B7- H3 (e).

In all experiments, DMSO was used as a vehicle control. All MTS data are average of three independent experiments ± S.E.M, normalized, and relative to untreated cells. Statistically significant results (p < .05) are marked with*

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cell line is more resistant to all targeted therapies tested and reinforces the potential benefit of B7- H3 inhibition in therapy- resistant tumors.

Simultaneous inhibition of MAPK and AKT/mTOR pathways did not give an additive antiproliferative effect in the HRAS^G12V mutated FEMX- 1 cells, which might be explained by cross talk between the two pathways. However, in BRAF^V600E mutated SKMEL- 28 cells, a

weak but significant enhanced growth inhibition upon treatment with combinations of vemurafenib and binimetinib with everolimus was observed (Figure 5). Interestingly, RAS mutations in melanomas have been found to activate both MAPK and PI3K/AKT/mTOR pathways, as opposed to BRAF that seems to only activate the MAPK pathway (Downward, 2003; Lasithiotakis et al., 2008). The cross talk between MAPK and PI3K/AKT/mTOR pathways in FEMX- 1 cells would then depend on both MEK and AKT activation as inhibition of both causes a similar antiproliferative effect. However, treatment with B7- H3 antibody or knockdown of B7- H3 increased the growth inhibition to each of the monotreatments, but not in combination treatment, indicating that B7- H3 exerts a similar effect on both pathways.

We did not observe significant differences in cell cycle distribution upon B7- H3 knockdown (data not shown). Of note, the reduced proliferation rate in B7- H3- inhibited and knockdown cells may be associated with their loss of glycolytic capacity. Glycolytic capacity has also been proposed to be a predictor of drug sensitivity in tumor models (Mookerjee, Nicholls, & Brand, 2016). It has been shown that B7- H3 suppresses Nrf2 activity, eventually leading to promotion of aerobic glycolysis (Lim et al., 2016). Thus, loss of B7- H3 may reduce cell proliferation and increase drug sensitivity through the inability to generate enough energy of growth.

The subcellular localization of B7- H3 could be important for its functional role in tumorigenesis, but its intracellular localization has not previously been addressed in detail in melanoma cells. Here, B7- H3 was found mainly in the cell membrane, but also in the cytoplasm of melanoma cells. Importantly, we present evidence that the cyto- plasmic localization of B7- H3 is within intracellular vesicles, that is, lysosomes and late endosomes, and that B7- H3 is present in extracellular vesicles. Exosomal localization has previously been reported only by mass spectrometry analyses of exosomes from melanoma cells (Lazar et al., 2015; Rappa, Mercapide, Anzanello, Pope, & Lorico, 2013). As the exosomal sorting of proteins is a tightly regulated pro- cess (Villarroya- Beltri, Baixauli, Gutierrez- Vazquez, Sanchez- Madrid,

& Mittelbrunn, 2014), our findings that exosomes from B7- H3- overexpressing melanoma cells have an increased expression of B7- H3 propose an active sorting of B7- H3 to these vesicles and that B7- H3 might act at a distance via exosomes. Also, this opens the pos- sibility of B7- H3 as a marker for detection of metastatic melanoma by non- invasive techniques.

Together, these results unveil a novel role for B7- H3 in melanoma sensitivity to chemotherapy and targeted therapy and support the hy- pothesis that targeting B7- H3 could be beneficial in metastatic melanoma treatment.

4 | METHODS

4.1 | Cell culture, plasmids, immunoblot, antibodies, and reagents

FEMX- 1 and HHMS cell lines were previously established from metastatic lesions of malignant melanoma patients treated at the Oslo University Hospital Radiumhospitalet (Fodstad et al., 1988;

Lillehammer et al., 2005). MeWo and SKMEL- 28 cells were purchased F I G U R E 5 Proliferation of SKMEL- 28 shB7- H3 cells treated with

BRAF, MEK, and mTOR inhibitors. Relative proliferation of the cells was measured by MTS assay after 3 days of treatment of SKMEL- 28 shSCR and shB7- H3 cells with 0.5 μ^m vemurafenib, 200 nm

everolimus, and combination of 0.5 μ^m vemurafenib and 200 nm

everolimus (a), or in SKMEL- 28 cell variants shSCR and shB7- H3 with 0.5 μ^m binimetinib, 200 nm everolimus, and combination of 0.5 μ^m binimetinib and 200 nm everolimus (b), or in parental SKMEL- 28 with or without 0.5 μ^m binimetinib, 1 μ^m vemurafenib, and 200 nm

everolimus with or without the presence of 100 ng/ml B7- H3 monoclonal inhibitory antibody (α- B7- H3, BRCA84D) (c). In all experiments, DMSO was used as a vehicle control. All MTS data are average of three independent experiments ± S.E.M., normalized, and relative to untreated cells. Statistically significant results (p < .05) are marked with*

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from ATCC. WM1366 and WM902b melanoma cells were kindly provided by Prof. M. Herlyn (Wistar Institute, Philadelphia, PA, USA).

All parental and variants of the melanoma cells were grown in RPMI- 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 2 m^m L- glutamine. BRAF^V600E mutant patient- derived cell lines (patients 1, 3, and 4) were established from biopsies before vemurafenib treatment as previously described (Lai, Jiang, Farrelly, Zhang,

& Hersey, 2012) and were a kind gift from Prof. P. Hersey (Kolling Medical Research Institute, Royal North Shore Hospital, University of Sydney, Australia). These were grown in DMEM (Invitrogen) supplemented with 2 m^m L- glutamine and 5% FBS (for patients 1 and 3) and 10% FBS (for Patient 4). Generation of shSCR and shB7- H3 cell variants was previously validated for specific knockdown as previously explained (Tekle et al., 2012). The mammalian expression plasmid to generate stable overexpressing cell lines was previously explained (Nunes- Xavier et al., 2016). Whole- cell protein extracts were pre- pared by total cell lysis, and immunoblot were performed as described previously (Nunes- Xavier et al., 2010; Nygren et al., 2014). Antibodies used for Western blotting were as follows: B7- H3 (AF1027, R&D), pERK1/2 (9101, Cell Signaling), ERK1/2 (sc- 93 and sc- 154, Santa Cruz), pAKT (4060, Cell signaling), AKT (9272, Cell Signaling), CD63 (ab59479, Abcam), tubulin (CP06, Millipore). B7- H3 inhibitory monoclonal antibody, BRCA84D, was kindly provided by MacroGenics, and 22F2 was from Dr. Reinhard Zeidler. Protein concentrations from total cell lysates were measured using The Pierce^® BCA Protein Assay Kit (Thermo Scientific, USA). Binimetinib (MEK- 162, MedChem Express), vemurafenib (PLX4032, Selleckchem), everolimus (RAD001,

InvivoGen), and triciribidine (API- 2, Sigma- Aldrich) were used at indicated concentration, during the indicated times. Dimethyl sulfox- ide (DMSO) was used as a control, at the same final concentration as resuspended and diluted drugs. Drug responses were plotted in GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA), and IC50 (half maximal inhibitory concentration) values were obtained for DTIC, vemurafenib, binimetinib, everolimus, and triciribidine in the cell variants and are presented in Table S1.

4.2 | Exosome purification and analysis

FEMX- 1 cells was plated in T160 flasks and grown in 20 ml RPMI- 1640 cell culture media with exosome- depleted FBS (GIBCO) for 3 days. Exosomes were purified by sequential centrifugation of cell culture supernatant, first by centrifugation at 1000 g for 5 min, then 2500 g for 10 min. To remove larger cell debris and possible apoptotic bodies, supernatant was centrifuged at 20.000 g for 20 min (Beckman Coulter, JA- 25.50 rotor). Finally, exosomes were collected by centrifugation at 100.000 g for 70 min (Beckman Coulter, 70Ti rotor) and washed in 10 ml PBS. Exosome fraction was verified by electron microscopy, lysed, and analyzed by immunoblot.

4.3 | In vitro proliferation, cell confluence, and colony formation assays

Cells (5 × 10³ cells) were plated in 96- well culture plates in media and treated with DMSO, DTIC, vemurafenib, binimetinib, everolimus, or F I G U R E 6 Effect of targeted therapy on patient- derived melanoma cell lines. (a) Immunoblot of B7- H3, pERK1/2, ERK1/2, pAKT, AKT, and tubulin expression from total lysates from patient- derived cell lines Patient 1, Patient 3, and Patient 4 established from biopsies. Plots show quantified immunoblot bands from B7- H3/tubulin, pERK1/2/ERK1/2, and pAKT/AKT in arbitrary units (A.U.) from three independent experiments ± S.E.M. (b, c) Cell confluence of Patient 1 (upper panel)- , Patient 3 (middle panel)- , and Patient 4 (lower panel)- derived melanoma cells was measured growing the cells in IncuCyte FLR or IncuCyte ZOOM Kinetic Imaging System (Essen BioScience) from one representative experiment ± S.D. in the presence of 100 ng/ml B7- H3 inhibitory antibody (α- B7- H3, BRCA84D) (b), or in combination with 3 μ^m vemurafenib, 1 μ^m binimetinib, and 200 nm everolimus (c). Bar graphs in C are based on the average end- point confluence of four or five independent experiments ± S.E.M. DMSO was used as a vehicle control. All cell confluence data were normalized, and relative to untreated cells. Statistically significant results (p < .05) are marked with*

(a)

(b) (c)

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triciribidine after 21 hr and processed after 72 hr post- treatment. Cell proliferation was determined by CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (MTS, Promega Corp, Madison, WI, USA).

Absorbance was measured at 490 nm using Wallac Victor2 1420 Multilabel Counter (PerkinElmer, USA). The data are presented as the average absorbance ± S.E.M corrected for background from at least three independent experiments. Cell confluence was measured growing the cells in IncuCyte FLR or IncuCyte ZOOM Kinetic Imaging System (Essen BioScience) that estimate cell growth and number. Cells were scanned every 3 hr during the times indicated. For colony formation assays, 500 cells were plated in six- well culture plates in media with DMSO or DTIC and processed after 14 days post- treatment.

Colonies were stained as described previously (Nunes- Xavier et al., 2010). The data are presented as cell confluence ± S.D. from one representative experiment. Assays were performed in at least triplicate wells three times for each cell line at separate days. Fold change in sensitivity ± S.E.M. for all compounds in the cell variants were quantified and are presented in Table S2.

4.4 | Extracellular acidification rate

XF96 glycolysis stress test was performed using Seahorse Extracellular Flux Analyzer XF96e to measure the extracellular acidification rate (ECAR) according to the manufacturer’s instructions. Cells were seeded in Seahorse plate 48 hr after splitting and cultured overnight to 80% confluence. Before measurement, the culture medium was replaced with cellular assay medium (Seahorse Bioscience) supplemented with 2 mm glutamine and incubated for 1 hr in a CO₂- free in- cubator. Assays were performed according to Seahorse protocols with the final concentrations of 10 mm glucose, 1 μ^m of oligomycin, and 100 mm of 2- deoxy- D- glucose (2- DG) and were performed in at least triplicate wells in three independent experiments for each cell line and condition at separate days ± S.D.

4.5 | Statistical analysis

Error bars in results represent data average ± standard deviation (S.D.) or standard error of the mean (S.E.M.) for results showing an average of indicated number of independent experiments. Two- tailed student t test was used to evaluate statistical significance. p values of p < .05 were considered significant and marked in the results with an asterisk.

ACKNOWLEDGEMENTS

We thank T. Øyjord for excellent technical assistance, Dr. E. Skarpen at the OUS- Montebello Advanced Light Microscopy Core Facility, Dr. S. Tveito for her expert assistance on exosome isolation, Prof. P.

Hersey for sharing of patient- derived cells, Prof. M. Herlyn for sharing cell lines, and MacroGenics and Prof. R. Zeidler for kindly providing B7- H3 monoclonal inhibitory antibodies. This work was supported by the following grants: KFK, CT, YA, ØF, CENX: The Norwegian Cancer Society, and CENX: The Research Council of Norway (grant number:

239,813). We also thank Gunnar Kristian Olsen and Randi Andresens legacy, and Arne E. Ingels’ legacy for financial support.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

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SUPPORTING INFORMATION

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How to cite this article: Flem-Karlsen K, Tekle C, Andersson Y, Flatmark K, Fodstad Ø, Nunes-Xavier CE. Immunoregulatory protein B7- H3 promotes growth and decreases sensitivity to therapy in metastatic melanoma cells. Pigment Cell Melanoma Res. 2017;00:1–10. https://doi.org/10.1111/pcmr.12599